A. Sampling and sample pre-processing, "Figure 1A":http://www.nature.com/protocolexchange/system/uploads/3349/original/Figure1.pdf?1414750643
A.1. Collect sample in a sterile tube and immediately snap-freeze it in liquid
nitrogen (on site). Immediate cryofixation of the sample is a key step in the experimental workflow to prevent any modification of the information contained within the DNA, RNA, protein and metabolite fractions. Biomolecular fractions are known to rapidly change due to specific and non-specific degradation, regulation of gene expression, and post-transcriptional and post-translational modifications.
A.2. Preserve the sample at -80 °C until pre-processing.
A.3. With a sterile spatula, reduce the sample to powder form (or very small pieces) around 200 mg +/- 10 % of frozen sample. To preserve the frozen state of the sample, cool down all equipment and refreeze regularly the material, by briefly dipping it into liquid nitrogen. Float a weighing boat on the surface of the liquid nitrogen and pour the powdered sample into this.
A.4. Transfer the frozen sample into a pre-cooled 2 ml microcentrifuge tube and refreeze this briefly in a liquid nitrogen bath. Optionally, the weighed sample can be stored for few days at -80 °C until further processing.
B. Extracellular metabolite extraction, "Figure 1B":http://www.nature.com/protocolexchange/system/uploads/3349/original/Figure1.pdf?1414750643
B.1. Thaw the sample on ice for approximately 10-15 min by placing the sample tube horizontally on the ice surface. Flip the tube regularly and verify the consistency of the sample every few minutes. Avoid further warming by placing the thawed sample tube in ice immediately once the sample is completely thawed.
B.2. Centrifuge the thawed samples at 18,000 × g for 15 min at 4 °C and transfer the supernatant into a fresh 2 ml sterile tube pre-cooled to 4 °C. If visible particles are still present in the supernatant, carry out an additional centrifugation for 5 min until a completely limpid supernatant is obtained. Proceed to step B.4. with the supernatant.
B.3. Snap-freeze the cell pellet and preserve it at -80 °C until step C.2.
B.4. Add to one volume of the aqueous supernatant obtained at step B.2. one volume (e.g. 150 μl) of cold methanol (at -20 °C) and two volumes (e.g. 300 μl) of cold chloroform (at -20 °C) and vortex.
B.5. Mix the supernatant and solvents by placing the tube in a shaker (Eppendorf ThermoMixer Comfort) set to maximum speed for 30 min at 4 °C.
B.6. Centrifuge the mixture at 14,000 × g and 4 °C for 5 min.
B.7. Transfer the polar (top) and non-polar (lower) phases into separate 2 ml tubes.
B.8. Aliquot the extracellular polar and non-polar metabolite fractions into glass vials (the volume to aliquot needs to be adjusted according to the analysis; we typically use a volume of 70 μl). Dry the polar metabolite fractions in a vacuum centrifuge and non-polar metabolite fractions under a chemical hood, cap tubes and preserve them at -80 °C until further analysis.
C. Intracellular metabolite extraction, "Figure 1C":http://www.nature.com/protocolexchange/system/uploads/3349/original/Figure1.pdf?1414750643
C.1. Cool down the nucleic acid-free (nucleic acids can be removed from milling balls e.g. by immersion in bleach) and autoclave-sterilized milling balls (5 × 2 mm + 2 × 5 mm) by dipping the 2 ml microcentrifuge tubes containing them in liquid nitrogen.
C.2. Add the cold millings balls to the previously obtained frozen cell pellet (step B.3.).
C.3. Cryomill the frozen pellet for 2 min at 25 Hz in a Retsch Mixer Mill MM 400 (Retsch). The adaptor rack holding the sample tubes needs to be pre-cooled in liquid nitrogen prior to the milling step. At the end of the cryomilling step, the sample should comprise a frozen homogenous powder.
C.4. Dip the tube immediately in liquid nitrogen to preserve the sample in a frozen state.
C.5. Sequentially add 300 μl of cold methanol:water (1:1; v/v) and 300 μl of cold chloroform. Vortex the mixture until complete dissolution of the pulverised sample in the solvent solution.
C.6. Mill the sample tube for an additional 2 min at 20 Hz in the Retsch Mixer Mill MM 400. In order to avoid solvent leakage, wrap Parafilm around the rim of the cap of the sample tube before starting the milling and remove it before the next step.
C.7. Centrifuge the sample tube at 14,000 × g and 4 °C for 5 min. This separates the solvent mixture into a polar (top) phase, an interphase pellet (middle) and non-polar (lower) phase.
C.8. Transfer the polar and non-polar phases into new 2 ml tubes.
C.9. Preserve the interphase with the milling beads in the original tube on ice and proceed immediately to section D.
C.10. Aliquot the intracellular polar and non-polar metabolites in glass vials as described in step B.8.
D. Cell lysis, "Figure 1D":http://www.nature.com/protocolexchange/system/uploads/3349/original/Figure1.pdf?1414750643
D.1. To prevent RNA degradation during cell lysis, add 10 μl of 2-β-mercaptoethanol to each ml of RLT buffer (Qiagen).
D.2. Add 600 µl of cold (4 °C) modified RLT buffer (step 1) to the interphase pellet (from step C.9.) and, to avoid leakage, cover the rim of the closed tube cap with Parafilm.
D.3. Resuspend the interphase in the modified lysis buffer by a quick vortexing of the sample tube.
D.4. Mill the sample for 30 sec at 25 Hz using the Retsch Mixer Mill MM 400. Ensure that the adaptor racks are pre-cooled to 4 °C before the milling step.
D.5. Transfer up to 700 μl of the lysate into a QIAshredder column (Qiagen) and centrifuge for 2 min at maximum speed and at room temperature. The entire lysate should pass through the QIAshredder column. If a pellet forms in the collection tube, it should be re-suspended before next step.
E. Biomacromolecular isolation and purification
E.1. All subsequent steps are summarized in "Figure 1E to 1G":http://www.nature.com/protocolexchange/system/uploads/3349/original/Figure1.pdf?1414750643 and described in detail in the AllPrep DNA/RNA/Protein Mini Handbook (Qiagen, version 09/2011), section “Simultaneous purification of genomic DNA, total RNA, and total protein from animal and human cells” (page 22 from step 4). In our own experience, the elution of DNA and total RNA in the dedicated buffer or water can be repeated in order to recover more nucleic acids (steps 13 and 24). All optional column-washing steps are routinely carried out in our laboratory.
F. Processing of bimolecular fractions
F.1. Check the quantity and quality of the RNA fraction, for example using the Agilent Bioanalyzer 2100 with the 6000 Nano RNA kit (Agilent; see “Anticipated Results” section).
F.2. Check the size and the quality (degraded versus intact) of the DNA fraction, for example by agarose gel electrophoresis (see “Anticipated Results” section).
F.3. Depending on the utilization of the biomolecular fractions, post-processing can be applied. We routinely carry out the following:
- quantification of the extracted biomacromolecules (DNA: Thermo Scientific NanoDrop 2000c; RNA: Agilent Bioanalyzer 2100 with the 6000 Nano RNA kit ; proteins: Life Technologies Qubit 2.0 fluorometer with the Qubit Protein Assay kit).
- ethanol precipitation of RNA followed by overlaying the RNA pellet with Ambion RNAlater solution and shipment to the sequencing facility on dry ice.
- TCA precipitation of the protein fraction.